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Histone deacetylase inhibitor SAHA induces ERa degradationin breast cancer MCF-7 cells by CHIP-mediated ubiquitinpathway and inhibits survival signaling
Xin Yi, Wei Wei, Sheng-Yu Wang, Zhi-Yan Du, Yuan-Ji Xu, Xiao-Dan Yu *
Department of Pathology, Institute of Basic Medical Sciences, 27 Taiping Road, Beijing 100850, China
a r t i c l e i n f o
Article history:
Received 12 August 2007
Accepted 31 October 2007
Keywords:
HDACi
SAHA
ERa
Breast cancer
CHIP
Hsp90
a b s t r a c t
Estrogen receptor a (ERa) plays an important role in the development and progression of
breast cancer, and recent studies showed that ERa expression is associated with resistance
to hormonal therapy. Therefore, a number of studies have explored ways to deplete ERa
from breast cancer cells as a new therapy especially for hormone-refractory breast cancer.
We reported here that suberoylanilide hydroxamic acid (SAHA), a histone deacetylase
inhibitor, effectively depletes ERa in breast cancer MCF-7 cells. However, the intrinsic
mechanisms by which SAHA decreases ERa levels are not clear. Our present data demon-
strated that both inhibition of ERa mRNA level and promotion of ERa degradation by the
proteasome contribute to SAHA-induced ERa depletion, indicating that SAHA may exert its
effects through transcriptional and posttranslational mechanisms. Furthermore, the
decrease of ERa protein level in MCF-7 cells after SAHA treatment is mainly the result of
its rapid degradation by the ubiquitin-proteasome pathway rather than transcriptional
inhibition. In addition, we showed that inactivation of the heat shock protein-90 (Hsp90) is
involved in SAHA-induced ERa degradation, and ubiquitin ligase CHIP (C-terminal Hsc70
interacting protein) enhances SAHA-induced ERa degradation. SAHA-induced ERa depletion
is paralleled with reduction of transcriptional activity of ERa and SAHA is able to effectively
inhibit cell proliferation and induce apoptosis of MCF-7 cells. Taken together, our results
revealed a mechanism for SAHA-induced ERa degradation and indicated that SAHA is a
ical agent for depletion of ERa and a potential choice for breast cancer
expressing high ERa.
suitable pharmacolog
# 2007 Elsevier Inc. All rights reserved.
1. Introduction
Breast cancer is the most common form of malignant disease
in woman worldwide. It has been reported that ERa plays a
critical role in the initiation and progression of breast cancer
because approximately 70% of primary breast cancers are ERa
positive [1,2]. As a result, ERa has become an important target
in the treatment of hormone-responsive breast cancer.
Unfortunately, most patients initially responding to anti-
* Corresponding author. Tel.: +86 10 6693 2372; fax: +86 10 6821 3039.E-mail address: [email protected] (X.-D. Yu).
0006-2952/$ – see front matter # 2007 Elsevier Inc. All rights reserveddoi:10.1016/j.bcp.2007.10.035
estrogen therapies, such as tamoxifen, which is a standard
component of front-line therapy for ERa positive breast
cancer, will eventually become resistant to those therapies
[3]. Aromatase inhibitors (AIs) are new drugs used for
endocrine treatment of post-menopausal breast cancer and
have demonstrated efficacy in patients with breast cancer
resistant to anti-estrogens. However, resistance to AIs has also
been observed. The potential mechanisms of endocrine
resistance are not fully understood, but evidence suggests
.
b i o c h e m i c a l p h a r m a c o l o g y 7 5 ( 2 0 0 8 ) 1 6 9 7 – 1 7 0 51698
that tamoxifen-resistant tumors regrowth may be associated
with ERa signaling. Moreover, complex interactions between
ERa and growth factor signaling pathways are also involved,
which is thought to be one of the determinants of endocrine
resistance [4–6]. Therefore, depletion of ERa from breast
cancer cells may be a particularly powerful approach to block
ERa signaling, especially ERa/growth factor crosstalk, pre-
venting the development of endocrine resistance ultimately.
Unliganded ERa, like other steroid hormone receptors, is
maintained in a ligand-binding competent conformation by
associating with various Hsp90-based chaperone complexes
[7]. Hsp90 inhibitors, such as geldanamycin (GA), can inhibit
Hsp90 molecular chaperone function and induce ERa degra-
dation through the ubiquitin-proteasome pathway [8,9].
Recently, it was found that carboxyl terminus of Hsc70-
interacting protein (CHIP), which is a co-chaperone of Hsp90/
Hsp70 complex, is involved in GA-induced ERa degradation as
an ubiquitin ligase [10]. However, anti-estrogen fulvestrant,
which also induces ERa degradation by dissociating the
complex of Hsp90 with ERa, reducing the interaction between
CHIP and ERa after treatment with GA, indicating that distinct
downstream pathways exist for ERa degradation by treatment
with different drugs [10]. Therefore, it is necessary for us to
elucidate the molecular mechanism of new drug-induced-ERa
degradation.
Histone deacetylase (HDAC) inhibitors, a promising class
of antitumor agents, can block the proliferation and induce
cell death in a wide variety of transformed cells [11].
However, the antitumor mechanism of HDAC inhibitors
has not been completely elucidated. It has been proved that
HDAC inhibitors selectively affect gene transcription by
acetylation of histones, such as SAHA and LAQ824, which
transcriptionally up-regulate p21 and increase p27 expres-
sion. This is associated with cell cycle arrest and apoptosis in
cancer cells, and is shown to induce in vivo regression of
tumors [12–14]. HDAC inhibitors-induced mitotic defect by
causing aberrant acetylation of histones in heterochromatin
and centromere domains per se can result in cell death by
either apoptosis or mitotic death/catastrophe [15]. It has also
been shown that SAHA can induce polyploidy and lead to cell
senescence in transformed cells [16]. These findings indi-
cated that HDAC inhibitors can cause tumor cell growth
arrest, apoptosis, mitotic cell death and polyploidy to exert
antitumor effects.
Recently, a number of studies showed that the antitumor
effects of HDAC inhibitors may be associated with Hsp90
acetylation induced by HDAC inhibitors, such as depsipeptide
(FK228), LAQ824 and SAHA. It has been proposed that Hsp90
acetylation correlates with inactivation of Hsp90 and induces
Hsp90 client proteins disassociation from Hsp90 molecular
complex, e.g. Raf-1, androgen receptor (AR) and HER2 [17–19].
An earlier research suggested that HDAC inhibitors deplete
ERa protein [20]. However, little is known regarding the
molecular mechanism of ERa depletion by HDAC inhibitors.
Suberoylanilide hydroxamic acid (SAHA) is an HDAC inhibitor,
which is in phase I/II clinical trials and has shown antitumor
activity in hematologic and solid tumors at doses well
tolerated by patients [21,22]. In present studies, we showed
that SAHA can suppress ERa mRNA level and induce ERa
degradation in breast cancer MCF-7 cells. The molecular
mechanism of SAHA-induced ERa degradation is associated
with inactivation of Hsp90, and co-chaperone CHIP of Hsp90 is
also involved as E3 ubiquitin ligase. The effect of SAHA on ERa
degradation is augmented by CHIP overexpression, but not
CHIP mutants’ overexpression, suggesting that CHIP partici-
pates in SAHA-induced ERa degradation. Most importantly,
SAHA-induced ERa depletion results in down-regulation of
ERa transcriptional activity and inhibition of cells proliferation
in MCF-7 cells. Therefore, SAHA may exert its antitumor effect
via depletion of ERa and inactivation of the crosstalk between
ERa and growth receptors signaling pathways.
2. Materials and methods
2.1. Cell, reagents and plasmids
The breast cancer cell line, MCF-7 cell line was obtained from
American Type Culture Collection (Manassas, VA, USA).
Suberoylanilide hydroxamic acid (SAHA) was offered by
AstraZeneca Company (Macclesfield, SK, UK). 17b-Estradiol,
4-hydroxytamoxifen, and MG-132 were purchased from Sigma
Chemical Company (St. Louis, MO, USA). ICI 182,780 was
purchased from Tocris Cookson Ltd. (Ellisville, MO, USA).
Antibodies for Raf-1, phospho-AKT, AKT, phospho-ERK, ERK,
acetylated-lysine, survivin, Myc, actin and ubiquitin were
obtained from Cell Signaling Technology (Beverly, MA, USA).
Anti-ERa and anti-CDK4 antibody was purchased from Santa
Cruz Biotechnology (Santa Cruz, CA, USA). Anti-Hsp90 anti-
body and anti-Hsp70 antibody were obtained from Stressgen
Biotechnologies Corporation (Victoria, BC, Canada). Anti-CHIP
antibody was purchased from Abcam Ltd. (Cambridge, UK).
Lipofectamine 2000 was obtained from Invitrogen Corporation
(Rockville, MD, USA). The Myc-CHIP (WT), Myc-CHIP (DTPR),
and Myc-CHIP (DU-box) constructs were kindly provided by Dr.
Yili Yang (NIH/NCI). ERE-pS2-Luc was kindly offered by Dr. Qi-
Nong Ye (Beijing Institute of Biotechnology, Beijing, China).
2.2. Cell culture and transient transfection
MCF-7 cells were cultured in DMEM medium containing 10%
fetal bovine serum at 37 8C in 5% CO2. Before experiments,
cells were cultured in hormone-free medium (phenol red-free
MEM with 5% charcoal-stripped FBS) for 3 days. For transient
transfection, cells were transfected with an equal amount of
total plasmid DNA by using Lipofectamine 2000 according to
the manufacturer’s guidelines.
2.3. Stable transfection
MCF-7 cells were transfected with pcDNA3.1-Myc-CHIP (WT),
pcDNA3.1-Myc-CHIP (DTPR), pcDNA3.1-Myc-CHIP (DU-box), or
empty vector by using Lipofectamine 2000 and selected in
growth medium containing 800 mg/ml G418 for 3 weeks. Then
drug-resistant colonies were chosen and expanded in growth
medium containing 300 mg/ml G418. The expression of Myc-
CHIP (WT), Myc-CHIP (DTPR), and Myc-CHIP (DU-box) in stable
cell lines (MCF-7) were detected by Western blot with anti-Myc
antibody. In parallel, several empty plasmid-transfected
clones were randomly selected and used as control cells.
b i o c h e m i c a l p h a r m a c o l o g y 7 5 ( 2 0 0 8 ) 1 6 9 7 – 1 7 0 5 1699
2.4. Western blot, immunoprecipitation analysis andluciferase assay
For Western blot analysis, control or SAHA-treated MCF-7
cells were lysed in Laemmli buffer (Bio-Rad Laboratories, CA,
USA), approximately 60 mg of total proteins were resolved on
SDS polyacrylamide gels and immunoblot was performed as
previously described [19]. For immunoprecipitation experi-
ments, cellular extracts from approximately 1 � 107 cells
were prepared in RIPA buffer, approximately 400 mg of total
proteins were used for immunoprecipitation analysis fol-
lowed by the procedure previously described [19]. For
luciferase assay, cell lysates were prepared and carried out
as recommended by Luciferase Assay System (Promega). Data
were expressed as mean � S.E.M. Statistical analysis was
performed by Student’s t-test and P values less than 0.05 were
considered significant.
2.5. RT-PCR
Total RNA was extracted from cultured cells with TRIzol
reagent according to the manufactured guidelines (Invitro-
gen). Reverse transcription (RT) was performed using First
Strand cDNA Synthesis Kit (Fermentas). Primer pairs used for
ERa PCR were as follows: forward, 50-TGATCCTACCA-
GACCCTTCA-30, and reverse, 50-TCCTGTCCAAGAGCAAGTT-
30. G3PDH was amplified and used as a standard for the PCR
reaction.
Fig. 1 – SAHA induces ERa degradation via the ubiquitin-protea
with different concentrations of SAHA for 24 h or treated with 5
the protein level of ERa was examined with anti-ERa antibody b
indicated times in the presence of proteasome inhibitor MG-13
extracted and mRNA level of ERa was detected by RT-PCR analy
treated with an inhibitor of protein synthesis, 100 mM cyclohexim
at indicated times. As a control, MCF-7 cells were treated with
protein level of ERa was detected by Western blot with anti-ERa
4 h in the presence of proteasome inhibitor MG-132 or in the ab
30 min before and continued during the SAHA treatment. Cell ly
with anti-ERa antibody, resolved by SDS-PAGE, and immunoblo
treated with SAHA (5 mM) at indicated times in the presence of
(5 mM). As a control, MCF-7 cells were treated with MG-132 (5 m
detected by Western blot with anti-ERa antibody.
2.6. Cell proliferation assay
MCF-7 cells were plated into 96-well plates at a density of
6 � 104/well and cultured in hormone-free medium. After 3
days, the medium was replaced with fresh medium in the
presence or absence of 1.25–10 mM SAHA. At appropriate time
points, the percentages of viable cells after treatment were
measured using the MTT assay. Each experiment was
performed three times.
3. Results
3.1. SAHA depletes ERa via the ubiquitin-proteasomepathway in MCF-7 cells
To investigate the effect of SAHA on ERa expression, we used
SAHA to treat breast cancer MCF-7 cells, which express high
ERa protein, the results showed that SAHA induces dose- and
time-dependent inhibition of ERa protein level (Fig. 1A).
Similar inhibition was observed with depsipeptide (FK228)
and trichostatin A (data not shown), which are type I and pan-
histone deacetylase inhibitors, respectively [20]. Previous
reports showed that HDAC inhibitors like LAQ824 exert their
effects on depleting androgen receptor through transcrip-
tional and posttranslational mechanisms [18]. To determine
whether SAHA-mediated ERa depletion is due to the same
mechanisms, we treated MCF-7 cells with SAHA for 6–24 h and
some pathway in MCF-7 cells. (A) MCF-7 cells were treated
mM SAHA at indicated times. Cell lysate was prepared and
y Western blot. (B) MCF-7 cells were treated with SAHA at
2 or in the absence of MG-132 (5 mM). Total RNA was
sis, G3PDH served as loading control. (C) MCF-7 cells were
ide (CHX) for 30 min, followed by addition of SAHA (5 mM)
100 mM cycloheximide (CHX) for 2, 4 and 6 h alone. The
antibody. (D) MCF-7 cells were treated with SAHA (5 mM) for
sence of MG-132 (5 mM). MG-132 was added to the cells
sate was lysed in RIPA buffer and immunoprecipitated (IP)
tted (IB) with anti-ubiquitin antibody. (E) MCF-7 cells were
proteasome inhibitor MG-132 or in the absence of MG-132
M) for 2, 4 and 6 h alone. The level of ERa protein was
b i o c h e m i c a l p h a r m a c o l o g y 7 5 ( 2 0 0 8 ) 1 6 9 7 – 1 7 0 51700
performed RT-PCR analysis to detect ERa mRNA level.
Comparison of the declining speed of ERa in protein and in
mRNA levels showed that, the remarkable decline of ERa
protein appeared at 6 h followed by the SAHA exposure,
whereas the faint decline of ERa in mRNA level appeared until
SAHA treatment for 12 h (Fig. 1B). The lagging of transcrip-
tional inhibition suggested that SAHA may decline ERa protein
level mainly via affecting its stability. To prove this hypoth-
esis, we treated MCF-7 cells for various times with SAHA, in
the presence or absence of cyclohexamide (CHX), an inhibitor
of protein synthesis, in the presence or absence of SAHA, and
then measured the relative ERa protein level in MCF-7 cells. As
shown in Fig. 1C, ERa protein level decreases faster in the cells
treated with SAHA in the presence of CHX than in the cells
treated with CHX alone. This result supported that inhibitory
effects of SAHA on ERa are mainly due to accelerate ERa
degradation in MCF-7 cells. To determine whether the
ubiquitin-proteasome system is responsible for SAHA-
induced ERa degradation, we treated MCF-7 cells with SAHA
at indicated times in the presence or absence of proteasome
inhibitor MG-132. ERa protein was immunoprecipitated and its
ubiquitination status was examined by Western blot with
anti-ubiquitin antibody. Fig. 1D shows that SAHA treatment
induces ubiquitination of ERa and this effect is evidently
enhanced by cotreatment of SAHA and MG-132. Furthermore,
Fig. 2 – SAHA acetylates Hsp90 and induces dissociation of ERa
proteins in MCF-7 cells. (A) SAHA induces Hsp90 acetylation. MC
lysed in RIPA buffer. Acetylated lysine was immunoprecipitated
PAGE, and immunoblotted (IB) with anti-Hsp90 or anti-ERa ant
loading control. (B) SAHA disassociates the binding between ERa
indicated times, cell lysate was lysed in RIPA buffer and immuno
PAGE, and immunoblotted (IB) with anti-Hsp90 or anti-ERa anti
as the loading control. (C) SAHA depletes Hsp90 client proteins.
the cell lysate from MCF-7 cells treated with SAHA (5 mM) for 6–
the proteasome inhibitor MG-132 prevents SAHA-induced ERa
degradation (Fig. 1E), suggesting that SAHA could induce ERa
degradation via the ubiquitin-proteasome pathway. Although
proteasome inhibitor MG-132 can completely prevent SAHA-
induced ERa protein degradation, it has no effect on SAHA-
induced decline on ERa mRNA level (Fig. 1B).
3.2. SAHA induces Hsp90 acetylation and Hsp90-ERacomplex disassociation in MCF-7 cells
It has been discovered that the association of ERa with the
heat shock protein-90 (Hsp90) molecular chaperone complex
is required for its stability and function [7]. Recently, it was
shown that HDAC inhibitors induce acetylation of Hsp90,
which inhibits its ATP binding and chaperone association with
its client proteins [17–19]. Consistent with these studies, we
next determined whether Hsp90 acetylation is involved in
SAHA-induced ERa depletion. Further experiments were then
performed to assess the effect of SAHA on acetylation of Hsp90
and ERa by immunoprecipitation with anti-acetylated lysine
antibody and immunoblotting with anti-Hsp90 or anti-ERa
antibodies, respectively. The results showed that treatment
with 5 mM SAHA as short as 2 h has already induced
acetylation of Hsp90 in MCF-7 cells, and the effect was
enhanced at 4 h but without significantly affecting the protein
from Hsp90 chaperone complex and depletes Hsp90 client
F-7 cells, treated with SAHA (5 mM) at indicated times, were
(IP) with anti-acetylated lysine antibody, resolved by SDS-
ibody. The level of Hsp90 in cell lysate was used as the
and Hsp90. MCF-7 cells were treated with SAHA (5 mM) at
precipitated (IP) using anti-ERa antibody, resolved by SDS-
body. The levels of Hsp90 and ERa in cell lysate were used
Western blot analyses of Raf-1, CDK4, survivin and actin in
48 h.
b i o c h e m i c a l p h a r m a c o l o g y 7 5 ( 2 0 0 8 ) 1 6 9 7 – 1 7 0 5 1701
level of Hsp90 in MCF-7 cells. Interestingly, in the same
conditions acetylation of ERa is not observed (Fig. 2A). Next, we
estimated the effect of SAHA on the association of Hsp90-ERa.
Following treatment with SAHA for 2 or 4 h, Hsp90-ERa
coimmunoprecipitation assay was done. As shown in Fig. 2B,
SAHA induces the disassociation of Hsp90 and ERa paralleled
by increased acetylation of Hsp90. These data implied that
SAHA induces Hsp90 acetylation and Hsp90-ERa disassocia-
tion in MCF-7 cells, resulting in ERa degradation via the
ubiquitin-proteasome pathway. Previous studies showed that
inactivation of Hsp90 molecular chaperone could induce
depletion of its client proteins such as AKT, Raf-1, and steroid
receptors [23–25]. We further evaluated the levels of Hsp90
client proteins such as Raf-1, CDK4 and survivin. Fig. 2C shows
that SAHA also mediates the depletion of these Hsp90 clients.
These results further indicated that SAHA-induced Hsp90
acetylation leads to disruption of its chaperone function and
decrease of its client proteins.
3.3. CHIP participates in SAHA-induced ERa degradationas an ubiquitin ligase
Previous studies showed that carboxyl-terminus of HSC70-
interecting protein (CHIP) is a U-box-containing E3 ubiquitin
ligase that binds through its tetratricopeptide repeat (TPR)
domain to independent TPR acceptor sites on Hsp90 and
Hsp70 [26,27]. CHIP has been shown to facilitate the
ubiquitination of Hsp90 client proteins, such as p53 and ErbB2
[28,29]. Most recently, CHIP has been reported to play a role in
GA-induced degradation of ERa [10]. Whether CHIP also plays a
role in SAHA-induced ERa degradation has not been investi-
gated. To determine whether CHIP participates in SAHA-
induced ERa degradation, we transfected wild type CHIP and
deficient mutant plasmids (DTPR and DU-box) into MCF-7 cells
and established the stable transfected cell lines. As a control,
MCF-7-Mock (MCF-7 cells stably transfected with empty
vector) was also established. We treated MCF-7-Mock cells
and MCF-7-CHIP (WT) with SAHA at indicated times. Fig. 3A
shows that ERa degradation is evidently accelerated in MCF-7-
CHIP (WT) cells compared with MCF-7-Mock cells. It has been
reported that CHIP associates with ERa and promotes ERa
ubiquitination in MCF-7 cells, we wonder to investigate the
function of CHIP in the case of SAHA-induced ERa degradation.
We treated MCF-7-CHIP (WT) cells with SAHA for 2 or 4 h, ERa
protein was immunoprecipitated with anti-ERa antibody, and
coimmunoprecipitated CHIP was detected by immunoblotting
with anti-Myc antibody. As shown in Fig. 3B, SAHA enhances
association of CHIP with ERa in a time-dependent manner. We
next examined whether CHIP could act as an ubiquitin ligase
mediating SAHA-induced ERa degradation. MCF-7-Mock cells
and MCF-7-CHIP (WT) cells were treated with SAHA for 2 h,
respectively, ubiquitination of ERa was slightly increased in
MCF-7-CHIP (WT) cells treated with SAHA (Fig. 3C), suggesting
a role for CHIP in SAHA-induced ERa ubiquitination. There-
fore, these results suggested that CHIP, by facilitating ERa
ubiquitination, targets ERa for proteasome-mediated degra-
dation. Furthermore, we evaluated whether overexpression of
the deletion mutant CHIP including CHIP (DTPR) and CHIP (DU-
box) could exert E3 ubiquitin ligase function in SAHA-induced
ERa degradation. As shown in Fig. 3D, overexpression of CHIP
(DU-box) and CHIP (DTPR) mutant, but not CHIP (WT), have no
effects on SAHA-induced ERa degradation. These data
indicated that intact CHIP is necessary to mediate SAHA-
induced ERa degradation.
3.4. SAHA down-regulates ERa transcriptional activityand blocks survival signaling in MCF-7 cells
We have confirmed that SAHA is capable to depleting ERa
expression through two mechanisms involving inhibition of
ERa mRNA level and induction of ERa degradation via CHIP-
mediated ubiquitin-proteasome pathway. To determine
whether SAHA-induced ERa depletion is associated with
attenuated ERa transcriptional activity, following treatment
with SAHA for 6 h, we transiently transfected MCF-7 cells with
an ERE-pS2-Luc reporter plasmid, then stimulated with E2 for
24 h and analyzed with luciferase assay. As shown in Fig. 4A,
SAHA decreases E2-induced ERa transcriptional activity in a
dose dependent manner accompanied with remarkably
decline of ERa protein level at same time. We assume that
the inhibition effect of SAHA on ERa transcriptional activity is
indirectly caused by ERa protein degradation, more experi-
ments using stable transfected cell lines, MCF-7-CHIP (WT),
MCF-7-CHIP (DTPR) and MCF-7-CHIP (DU-box) further demon-
strated that SAHA causes a stronger decrease of ERa
transcriptional activity inMCF-7-CHIP (WT) cells but not in
MCF-7-CHIP (DTPR) and MCF-7-CHIP (DU-box) cells, which is
consistent with its effect on depleting ERa from different cell
lines (Fig. 3D).
We further evaluated the effects of SAHA on survival
signaling pathways in MCF-7 cells. As shown in Fig. 4B,
treatment with SAHA resulted in the reduction of phosphory-
lated ERK or phosphorylated AKT in a time-dependent
manner, indicating that ERK and AKT activities are inhibited
in these cells. Coincidence with the inhibition of ERK and AKT
activities, the level of cleaved-PARP was increased, suggesting
that inhibition of the Raf-1/MEK/ERK and PI3K/AKT survival
signaling pathways is involved in the SAHA-induced apopto-
sis. Next, MCF-7 stable transfected cells were treated with
SAHA for 24 h; the result showed that CHIP overexpression
enhances the effect of SAHA on inactivating survival signaling
pathways (Fig. 4B).
We have showed that treatment with 5 mM SAHA is able to
induce ERa degradation and inhibit ERa transcriptional
activity. To examine the growth inhibitory effect of SAHA in
MCF-7 cells, MCF-7 cells were treated with SAHA for 0–72 h.
MTT assay demonstrated that SAHA causes dose and time-
dependent decrease in cell viability. The viability of MCF-7
cells was inhibited by 60% after 72 h, suggesting that SAHA is
effective in inhibiting MCF-7 cell proliferation (Fig. 4C).
Furthermore, to evaluate the correlation between SAHA-
induced cytotoxicity and ERa depletion in MCF-7 cells, we
compared the growth inhibitory effect of SAHA on MCF-7 cells
with that on MCF-7-CHIP (WT), MCF-7-CHIP (DTPR) and MCF-7
(DU-box) cells. The result showed that the cytotoxicity of SAHA
is obviously enhanced in MCF-7-CHIP (WT) cells, whereas no
difference among MCF-7 cells, MCF-7-CHIP (DTPR) and MCF-7-
CHIP (DU-box) cells. It is indicated that promoting ERa
depletion by CHIP overexpression could enhance SAHA
cytotoxicity (Fig. 4C). Taken together, SAHA down-regulates
Fig. 3 – CHIP is required for SAHA-induced ERa degradation in MCF-7 cells. (A) Overexpression of CHIP promotes SAHA-
induced ERa degradation. MCF-7 cells or MCF-7-CHIP (WT) cells were treated with SAHA (5 mM) at indicated times, cell
lysate was prepared and the protein levels of ERa and CHIP were detected by Western blot with anti-ERa and anti-Myc
antibody, respectively. (B) SAHA enhances CHIP-ERa interaction. MCF-7-CHIP (WT) cells were treated with SAHA (5 mM) at
indicated times, and cell lysate was lysed in RIPA buffer and immunoprecipitated (IP) using anti-ERa antibody, resolved by
SDS-PAGE, and immunoblotted (IB) with anti-ERa or anti-Myc antibody. (C) CHIP enhances SAHA-induced ERa
ubiquitination. Immunoprecipitation (IP) was performed with anti-ERa antibody and immunoblotted (IB) with anti-
ubiquitin, anti-ERa and anti-Myc antibody, respectively in the RIPA buffer from MCF-7 or MCF-7-CHIP (WT) cells treated
with SAHA (5 mM) for 2 h. The level of ERa in cell lysate was used as the loading control. (D) CHIP’s function is dependent on
both TPR and U-box domain. SAHA (5 mM) treated these stable transfected cells (MCF-7-Mock, MCF-7-CHIP (WT), MCF-7-
CHIP (DTPR) and MCF-7-CHIP (DU-box)) for 6 h. The expression of exogenesis CHIP and its mutants were detected by anti-
Myc antibody. ERa protein was detected by Western blot with anti-ERa.
b i o c h e m i c a l p h a r m a c o l o g y 7 5 ( 2 0 0 8 ) 1 6 9 7 – 1 7 0 51702
ERa transcriptional activity and blocks survival signaling in
MCF-7 cells via CHIP-mediated mechanism.
4. Discussion
It has been proposed that levels of ERa are commonly
increased in premalignant and malignant breast lesions,
and ERa plays a crucial role for the growth and survival of
breast cancer cells. Recently, accumulated evidences also
have shown that complex interactions between ERa and
growth factor signaling pathways are one of the potential
mechanisms of endocrine resistance to breast cancer [4–6].
Thus, ERa becomes an important target for the treatment of
breast cancer. Depletion of ERa protein by Hsp90 inhibitors or
ERa-small interfering RNA (siRNA) has been shown to
suppress breast tumor growth in vivo [8,9,30]. Therefore,
depletion of ERa from breast cancer cells is a rational
therapeutic strategy not only for primary estrogen-dependent
breast cancer but also for hormone-refractory breast cancer.
Our experiments confirmed that SAHA, a HDAC inhibitor,
currently in human clinical trials, is able to deplete ERa in
breast cancer MCF-7 cells.
Our data showed that SAHA depletes ERa through two
mechanisms: attenuation of its mRNA level and promotion
of its degradation by ubiquitin-proteasome pathway. It is
similar to what has been previously observed that HDAC
inhibitor LAQ824 and TSA deplete HER2, Bcr-Abl, and AR
through transcriptional and posttranslational mechanisms.
It seems that HDAC inhibitors-induced degradation of
oncoproteins maybe commonly accompanied by down-
regulation of their mRNA levels. However, recent report
showed that TSA has no effect on the levels of HIF-1a mRNA
transcripts while it induces rapid degradation of HIF-1a
protein [31], suggesting that the effect of HDAC inhibitors is
different for various types of Hsp90 client proteins. Our
current study has focused on the molecular mechanism of
SAHA-induced ERa degradation. Previous report showed
that TSA, another HDAC inhibitor, induced proteasome-
independent down-regulation of ERa [32]. However, in this
study, we observed that MG-132, a proteasome inhibitor,
could effectively inhibit SAHA-induced ERa degradation and
enhance ERa ubiquitination. Therefore, our data indicated
that SAHA induces ERa degradation via the ubiquitin-
proteasome pathway. The basis for these disparate results
is not clear, but may reflect different effects of TSA versus
Fig. 4 – SAHA down-regulates ERa transcriptional activity and inactivates survival signaling in MCF-7 cells. (A) After
treatment with SAHA (5–10 mM) for 6 h, ERE-Luc plasmid (250 ng) was transiently transfected into MCF-7 cells and the stable
transfected cells (MCF-7-CHIP (WT), MCF-7-CHIP (DTPR) and MCF-7-CHIP (DU-box)) for 24 h; cells were treated with 10 nM E2
or DMSO as indicated and assayed 24 h later for relative luciferase activity. P < 0.01 (student’s t-test, vs. first group) is
indicated by a *. ERa protein was detected by Western blot with anti-ERa. (B) Left figure: MCF-7 cells were treated with SAHA
(5 mM) at indicated times. Right figure: MCF-7 cells and the stable transfected cells (MCF-7-CHIP (WT), MCF-7-CHIP (DTPR)
and MCF-7-CHIP (DU-box)) were treated with SAHA (5 mM) for 48 h. Cell lysate was analyzed by Western blot with
antibodies to p-AKT, AKT, p-ERK, ERK, PARP, Myc or actin. (C) Upper figure: MCF-7 cells were treated with SAHA at 1.25–
10 mM at indicated times. Lower figure: MCF-7 cells and the stable transfected cells (MCF-7-CHIP (WT), MCF-7-CHIP (DTPR)
and MCF-7-CHIP (DU-box)) were treated with SAHA at 1.25–10 mM for 48 h. Cytotoxicity was evaluated by MTT assays. All
experiments were performed in triplicate.
b i o c h e m i c a l p h a r m a c o l o g y 7 5 ( 2 0 0 8 ) 1 6 9 7 – 1 7 0 5 1703
SAHA or attribute to the different doses and times of MG-132
used in the studies.
Recently it has been showed that HDAC inhibitors such as
LAQ824, FK228 and SAHA exert their anti-cancer effects by
inactivating of Hsp90 molecular chaperone function, impair
the chaperone association of Hsp90 with its client proteins,
HER2, AR and induce the degradation via the ubiquitin-
proteasome pathway. In our studies, the results indicated that
SAHA-induced ERa degradation in MCF-7 cells is also
associated with inactivation of Hsp90 molecular chaperone
function. First, treatment with SAHA induces Hsp90 acetyla-
tion, and Hsp90 acetylation is linked to the inactivation of its
chaperone function. The disassociation of ERa from the Hsp90
chaperone complex is accompanied with SAHA-induced
acetylation of Hsp90. Secondly, besides ERa, other Hsp90
client proteins, such as Raf-1, CDK4 and survivin are also
reduced by SAHA. Therefore, these observations suggested
that SAHA induces ERa degradation via the ubiquitin-protea-
some pathway by inhibiting Hsp90 chaperone function in
breast cancer MCF-7 cells. Previous reports have shown that
treatment with SAHA induces p21 and cell cycle growth arrest,
associated with differentiation and apoptosis of breast cancer
cells [13]. Our data indicated that antitumor effects of SAHA
are also associated with inhibition of Hsp90 function, which
leads to Hsp90 client proteins’ degradation.
It is known that molecular chaperones recognize non-
native proteins and aid in their correct folding. When it is
unsuccessful, the misfolded proteins will be directed to
degradation. The mutually exclusive pathways of folding
and degradation constitute the cell’s protein quality control
b i o c h e m i c a l p h a r m a c o l o g y 7 5 ( 2 0 0 8 ) 1 6 9 7 – 1 7 0 51704
system [33]. As recently studies showed, another co-chaper-
one, CHIP, regulates chaperone function in part by regulating
ubiquitin-proteasome pathway and determining whether
proteins enter the productive folding pathway or the degrada-
tion pathway. CHIP acts as an E3 ubiquitin ligase and promotes
ubiquitination of Hsp90 client proteins by a COOH-terminal U-
box domain, while binding molecular chaperone Hsp90 or
Hsp70 through TPR domain [34]. Recently it has been reported
that ERa serves as a substrate for Hsp90/Hsp70-associated
CHIP. The association of CHIP with ERa through a chaperone
intermediately results in ubiquitination and degradation of
ERa. And the presence of Hsp90 inhibitor GA potently
stimulates this process. Our data demonstrated that E3
ubiquitin ligase CHIP also mediates SAHA-induced ERa
ubiquitination and proteasomal degradation. CHIP overex-
pression in the presence of SAHA leads to an additive loss of
ERa and decreases its transcriptional activity via enhanced
ERa ubiquitination. The accelerate degradation of ERa further
mediates inactivation of survival signaling pathways and
enhances SAHA-induced cytotoxicity. The function of CHIP on
mediating ERa degradation is dependent on both the TPR
domain and the U-box E3 ligase activity, as transfection with
either CHIP (DTPR) or CHIP (DU-box) has no effect on SAHA-
induced ERa degradation or cytotoxicity. Taken together, we
concluded that E3 ubiquitin ligase CHIP is necessary for ERa
degradation, following Hsp90 inhibition by SAHA. These
findings are consistent with previous studies, which showed
that overexpression of CHIP by transient transfection
enhances ERa degradation [10].
Although tamoxifen has been the standard endocrine
treatment for breast cancer for many years, its extended use
may be associated with the development of tamoxifen
resistance [3]. The potential mechanisms of endocrine
resistance are not fully clarified, but evidence suggested that
complex interactions between ERa and growth factor signaling
pathways are involved. The introduction of new endocrine
agents that can induce ERa depletion and block growth
signaling pathways, may improve the therapeutic options for
women with endocrine-resistant breast cancer. Here, we
demonstrated that SAHA, a HDAC inhibitor, induces rapid
depletion of ERa protein, resulting in repression of the ERa-
dependent transcriptional function. Consistent with our
results, Reid and his colleagues reported that another HDAC
inhibitor, TSA, has the similar effect on influencing estrogen-
dependent transcription [35]. However, their data showed that
TSA-induced ERa clearance is due to reduction of the steady-
state level of ERa mRNA in MCF-7 cells and consequently
suppress the transactivation activity of ERa. In contrast, our
results showed at 6 h of SAHA treatment, the protein level of
ERa decreased markedly accompanied with inhibition of ERa-
dependent transcriptional activation, but with slight down-
regulation of ERa mRNA level, suggesting that SAHA-induced
ERa transcriptional inactivation is mainly caused by ERa
degradation, but not reduction of ERa mRNA. Our results
implied that inhibition of ERa-dependent transcriptional
activation by SAHA could provide a potentially important
strategy to develop new therapeutic agents against breast
cancer. Activation of EGFR and HER2 receptors results in
stimulation of several signaling pathways including mitogen-
activated protein kinase (MAPK) and PI3K/AKT pathways. It
has been reported that ERa can be phosphorylated and
activated by these protein kinases; increased bidirectional
crosstalk between ERa and EGFR signaling pathways is
thought to contribute to the development of tamoxifen
resistance [6]. In our study, we demonstrated that SAHA
could reduce MAPK and PI3K/AKT signaling activity and
inhibit the growth of MCF-7 cells. Our data indicated that the
use of agents such as SAHA to degrade ERa protein level and
abrogate survival signaling pathways may lead to improve-
ments in the prevention of endocrine resistance. Our findings
also indicated that CHIP can mediate down-regulation of ERa
transcriptional activity and inhibition of survival signaling in
MCF-7 cells caused by SAHA, which implied that CHIP may
play an important role in HDAC inhibitors-induced antitumor
effects.
Acknowledgements
We thank Dr. Yili Yang and Dr. Qi-Nong Ye for providing
plasmids. This work was supported by the National Natural
Science Foundation of China (Grant Nos. 30330620 and
30470898).
r e f e r e n c e s
[1] Allred DC, Mohsin SK. Biological features of premalignantdisease in the human breast. J Mammary Gland BiolNeoplasia 2000;5(4):351–64.
[2] Harvey JM, Clark GM, Osborne CK, Allred DC. Estrogenreceptor status by immunohistochemistry is superior to theligand-binding assay for predicting response to adjuvantendocrine therapy in breast cancer. J Clin Oncol1999;17(5):1474–81.
[3] Ali S, Coombes RC. Endocrine-responsive breast cancer andstrategies for combating resistance. Nat Rev Cancer2002;2(2):101–12.
[4] Lonning PE. Cross-resistance to different aromataseinhibitors in breast cancer treatment. Endocr Relat Cancer1999;6(2):251–7.
[5] Gutierrez MC, Detre S, Johnston S, Mohsin SK, Shou J, AllredDC, et al. Molecular changes in tamoxifen-resistant breastcancer: relationship between estrogen receptor, HER-2, andp38 mitogen-activated protein kinase. J Clin Oncol2005;23(11):2469–76.
[6] Levin ER. Bidirectional signaling between the estrogenreceptor and the epidermal growth factor receptor. MolEndocrinol 2003;17(3):309–17.
[7] Pratt WB, Toft DO. Steroid receptor interactions with heatshock protein and immunophilin chaperones. Endocr Rev1997;18(3):306–60.
[8] Beliakoff J, Bagatell R, Paine-Murrieta G, Taylor CW,Lykkesfeldt AE, Whitesell L. Hormone-refractory breastcancer remains sensitive to the antitumor activity of heatshock protein 90 inhibitors. Clin Cancer Res2003;9(13):4961–71.
[9] Bagatell R, Khan O, Paine-Murrieta G, Taylor CW, Akinaga S,Whitesell L. Destabilization of steroid receptors by heatshock protein 90-binding drugs: a ligand-independentapproach to hormonal therapy of breast cancer. ClinCancer Res 2001;7(7):2076–84.
[10] Fan M, Park A, Nephew KP. CHIP (carboxyl terminus ofHsc70-interacting protein) promotes basal and
b i o c h e m i c a l p h a r m a c o l o g y 7 5 ( 2 0 0 8 ) 1 6 9 7 – 1 7 0 5 1705
geldanamycin-induced degradation of estrogen receptor-alpha. Mol Endocrinol 2005;19(12):2901–14.
[11] Marks PA, Richon VM, Miller T, Kelly WK. Histonedeacetylase inhibitors. Adv Cancer Res 2004;91:137–68.
[12] Johnstone RW, Licht JD. Histone deacetylase inhibitors incancer therapy: is transcription the primary target? CancerCell 2003;4(1):13–8.
[13] Richon VM, Sandhoff TW, Rifkind RA, Marks PA. Histonedeacetylase inhibitor selectively induces p21WAF1expression and gene-associated histone acetylation. ProcNatl Acad Sci USA 2000;97(18):10014–9.
[14] Fuino L, Bali P, Wittmann S, Donapaty S, Guo F, YamaguchiH, et al. Histone deacetylase inhibitor LAQ824 down-regulates Her-2 and sensitizes human breast cancer cells totrastuzumab, taxotere, gemcitabine, and epothilone B. MolCancer Ther 2003;2(10):971–84.
[15] Cimini D, Mattiuzzo M, Torosantucci L, Degrassi F. Histonehyperacetylation in mitosis prevents sister chromatidseparation and produces chromosome segregation defects.Mol Biol Cell 2003;14(9):3821–33.
[16] Xu WS, Perez G, Ngo L, Gui CY, Marks PA. Induction ofpolyploidy by histone deacetylase inhibitor: a pathway forantitumor effects. Cancer Res 2005;65(17):7832–9.
[17] Yu X, Guo ZS, Marcu MG, Neckers L, Nguyen DM, Chen GA,et al. Modulation of p53, ErbB1, ErbB2, and Raf-1 expressionin lung cancer cells by depsipeptide FR901228. J Natl CancerInst 2002;94(7):504–13.
[18] Chen L, Meng S, Wang H, Bali P, Bai W, Li B, et al. Chemicalablation of androgen receptor in prostate cancer cells bythe histone deacetylase inhibitor LAQ824. Mol Cancer Ther2005;4(9):1311–9.
[19] Bali P, Pranpat M, Swaby R, Fiskus W, Yamaguchi H, BalasisM, et al. Activity of suberoylanilide hydroxamic Acidagainst human breast cancer cells with amplification ofher-2. Clin Cancer Res 2005;11(17):6382–9.
[20] Margueron R, Duong V, Bonnet S, Escande A, Vignon F,Balaguer P, et al. Histone deacetylase inhibition andestrogen receptor alpha levels modulate the transcriptionalactivity of partial antiestrogens. J Mol Endocrinol2004;32(2):583–94.
[21] Kelly WK, Richon VM, O’Connor O, Curley T, MacGregor-Curtelli B, Tong W, et al. Phase I clinical trial of histonedeacetylase inhibitor: suberoylanilide hydroxamic acidadministered intravenously. Clin Cancer Res 2003;9(10 Pt1):3578–88.
[22] Kelly WK, O’Connor OA, Krug LM, Chiao JH, Heaney M,Curley T, et al. Phase I study of an oral histone deacetylaseinhibitor, suberoylanilide hydroxamic acid, in patients withadvanced cancer. J Clin Oncol 2005;23(17):3923–31.
[23] Fortugno P, Beltrami E, Plescia J, Fontana J, Pradhan D,Marchisio PC, et al. Regulation of survivin function byHsp90. Proc Natl Acad Sci USA 2003;100(24):13791–6.
[24] Schulte TW, Blagosklonny MV, Ingui C, Neckers L.Disruption of the Raf-1-Hsp90 molecular complex results indestabilization of Raf-1 and loss of Raf-1-Ras association. JBiol Chem 1995;270(41):24585–8.
[25] Stepanova L, Leng X, Parker SB, Harper JW. Mammalianp50Cdc37 is a protein kinase-targeting subunit of Hsp90 thatbinds and stabilizes Cdk4. Genes Dev 1996;10(12):1491–502.
[26] Connell P, Ballinger CA, Jiang J, Wu Y, Thompson LJ,Hohfeld J, et al. The co-chaperone CHIP regulates proteintriage decisions mediated by heat-shock proteins. Nat CellBiol 2001;3(1):93–6.
[27] Cyr DM, Hohfeld J, Patterson C. Protein quality control: U-box-containing E3 ubiquitin ligases join the fold. TrendsBiochem Sci 2002;27(7):368–75.
[28] Esser C, Scheffner M, Hohfeld J. The chaperone-associatedubiquitin ligase CHIP is able to target p53 for proteasomaldegradation. J Biol Chem 2005;280(29):27443–8.
[29] Zhou P, Fernandes N, Dodge IL, Reddi AL, Rao N, Safran H,et al. ErbB2 degradation mediated by the co-chaperoneprotein CHIP. J Biol Chem 2003;278(16):13829–37.
[30] Fu HJ, Jia LT, Bao W, Zhao J, Meng YL, Wang CJ, et al. Stableknockdown of estrogen receptor alpha by vector-basedRNA interference suppresses proliferation and enhancesapoptosis in breast cancer cells. Cancer Biol Ther2006;5(7):842–7.
[31] Kong X, Lin Z, Liang D, Fath D, Sang N, Caro J. Histonedeacetylase inhibitors induce VHL and ubiquitin-independent proteasomal degradation of hypoxia-inducible factor 1 alpha. Mol Cell Biol 2006;26(6):2019–28.
[32] Alao JP, Lam EW, Ali S, Buluwela L, Bordogna W, Lockey P,et al. Histone deacetylase inhibitor trichostatin A repressesestrogen receptor alpha-dependent transcription andpromotes proteasomal degradation of cyclin D1 in humanbreast carcinoma cell lines. Clin Cancer Res2004;10(23):8094–104.
[33] Wickner S, Maurizi MR, Gottesman S. Posttranslationalquality control: folding, refolding, and degrading proteins.Science 1999;286(5446):1888–93.
[34] McDonough H, Patterson C. CHIP: a link between thechaperone and proteasome systems. Cell StressChaperones 2003;8(4):303–8.
[35] Reid G, Metivier R, Lin CY, Denger S, Ibberson D, Ivacevic T,et al. Multiple mechanisms induce transcriptional silencingof a subset of genes, including oestrogen receptor alpha, inresponse to deacetylase inhibition by valproic acid andtrichostatin A. Oncogene 2005;24(31):4894–907.
